Fibronectin (FN) is an indispensable part of virtually all tissues and an important link in cell recognition of environmental signals. In tissues, FN functions from within a network of matrix fibrils that are assembled in a cell-mediated process. FN is also a functional component of the fibrin provisional matrix found in wounds. The primary goals of this proposal are to determine the molecular mechanisms of cell-mediated FN fibril formation, to define effects of changes in FN-fibrin matrix composition, and to develop systems for analyzing FN assembly and function in complex environments. To address the mechanisms and regulation of cell-mediated FN fibril assembly, we primarily use cell lines that lack an endogenous FN matrix in combination with normal and mutant recombinant FNs (recFNs) expressed using the baculovirus expression system. FN repeats III1-7 have been shown to play a regulatory role. Potential effects of this region on FN conformational changes and FN-FN interactions during assembly will be tested using solid phase and solution binding assays and cell assembly of recFNs with specific mutations. Contributions of actin stress fibers to this process will be determined using agents that induce or inhibit actin organization and cell contraction (such as LPA or actin-myosin inhibitors). FN-fibrin matrices present a tractable 3-dimensional system for analyzing cell-FN interactions in response to changes in matrix composition. Incorporation of a single additional protein into a FN-fibrin matrix causes a dramatic change in fibroblast morphology with many filopodia. Contributions to this change from integrins, actin binding proteins, and the Rho GTPase family will be probed using microscopic and biochemical approaches as well as cells expressing dominant Rho proteins. In vivo, FN matrices are assembled in complex environments. This will be modeled by following FN assembly by cells within 3-dimensional FN-fibrin matrices. To determine the requirements for regulation of FN assembly in tissues, embryonic stem cells with FN mutations that affect fibril formation will be used for in vitro differentiation and to generate FN mutations in vivo. This cohesive framework for characterizing the mechanisms and regulation of FN fibril formation and function will provide novel information relevant to many biological processes.